The patch clamp technique (Hamill, Neher, Sakmann and Sigworth, Improved Patch Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches, PFLUGERS ARCH. 391, 85-100, 1981) is used in the study of physiology, particularly in the study of behavior of ion channels and macroscopic currents in cells. The technique allows voltage clamped measurement of ionic current in either a small patch of cell membrane, or the entire membrane area of a small cell. Patch clamp studies are commonly used to facilitate drug screening in the pharmaceutical industry, particularly in drugs that act by blocking or regulating channel activity to and from cells, to directly assay the function of genes that encode ion channel and transporter proteins, and in neuroscience research, as well as various other applications.
In conventional patch clamp recording, a glass or fused-quartz micropipette having a tip opening on the order of 1 micron (1 μm) in diameter is gently applied to form a seal against a cell membrane, isolating a patch of the membrane with a seal resistance on the order of 1-100 GΩ. The micropipette is typically filled with saline solution, and acts as an electrode to permit detection and recording of ion channel current through the membrane, as well as observation and recording of the opening and closing of ion channels in the membrane.
Known methods of patch clamp recording, however, typically require a high degree of technical proficiency, and are quite time consuming and expensive. For example, the micropipette generally must be manually manipulated with extreme precision to contact and seal against a target cell under observation. And because known patch clamp methods analyze only a single cell or a patch of a cell's membrane surface, multiple patch clamp analyses typically must be carried out for statistical confirmation of the observations. But extreme difficulty in exactly replicating the study conditions over multiple procedures can drastically increase the expense and time required to complete a study, and add to the uncertainty of the observed results.
The application of a voltage across a biological or synthetic membrane can be utilized in a variety of applications. For example, application of a localized voltage across a membrane can be used to address or map biological structures such as ion channels and/or to detect binding events at a channel.
The presence or absence of such ion channels or carriers in a membrane can act as a molecular switching element that converts a binding event into an electrical signal, functioning as a transducer in a biosensor or nanodevice. For example, in a membrane in which a molecular channel or switch is held open when a specific analyte is bound, ion transport through the membrane is permitted when the analyte is bound, but is blocked when the analyte is not bound. If a voltage is applied across the membrane, a current pulse will be observed if ion transport occurs through the membrane, indicating an open channel and thus the presence of a binding event. Conversely, if a voltage is applied across the membrane and no current is observed (e.g., no ion transport through the membrane), a closed channel (and thus the absence of a binding event) is indicated.
The very small scale of the membranes and the molecules forming ion channel and ion carrier molecular switches under investigation (commonly on the order of about 100 Angstroms), as well as the relatively high density of ion channels on a substrate renders the addressing of these channels very difficult using known techniques. One conventional solution for the addressing of biological structures such as ion channels would be to make electrical connections to all or to many of these molecular switches. The applied voltage and responses of individual addresses on a substrate such as a silicon wafer surface could be scanned with the aid of computerized circuitry. However, the resolution of known addressable electrodes is poor, and manufacturing of an electrode system on the substrate surface would likely prove difficult and expensive. Also, voltage applied to a membrane in an electrolytic solution is typically conducted through the electrolyte along the membrane surface, rendering it difficult or impossible to address or map a specific location on the membrane.
Thus, it can be seen that needs exist for improved methods of and apparatus for patch clamp analysis. It is to the provision of improved apparatus and methods meeting this and other needs that the present invention is primarily directed.